EFFICACY OF DIETARY SODIUM BENTONITE AGAINST SUBCHRONIC EXPOSURE TO DIETARY AFLATOXIN IN BROILERS

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1 Bull Vet Inst Pulawy 50, , 2006 EFFICACY OF DIETARY SODIUM BENTONITE AGAINST SUBCHRONIC EXPOSURE TO DIETARY AFLATOXIN IN BROILERS GOKHAN ERASLAN, DINC ESSIZ 1, MEHMET AKDOGAN 3, ERDAL KARAOZ 4, MERAL ONCU 4 AND ZAFER OZYILDIZ 2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Erciyes, 38090, Kayseri, Turkey 1 Department of Pharmacology and Toxicology, 2 Department of Pathology, Faculty of Veterinary Medicine, University of Kafkas, 36100, Kars, Turkey 3 Department of Biochemistry, 4 Department of Histology and Embryology, Faculty of Medicine, University of Suleyman Demirel, 32260, Isparta, Turkey geraslan38@hotmail.com Received for publication October 14, Abstract Seventy-two one-day-old male broiler chicks were used in the study. The chicks were divided into six groups. The first group for 45 d was fed normal feed; at the same time the second, third, fourth, fifth and sixth groups were fed the feed containing 0.25% sodium bentonite, 0.50% sodium bentonite, 1 ppm of aflatoxin (approximately 85%, 10%, 3%, 2% of aflatoxins B 1, B 2, G 1, and G 2, respectively), 0.25% sodium bentonite+1 ppm of aflatoxin, and 0.50% sodium bentonite+1ppm of aflatoxin, respectively. On day 45, the animals were sacrificed and blood samples were taken in order to measure total protein, albumin, glucose, triglyceride, cholesterol, high-density lipoprotein, low-density lipoprotein, very low-density lipoprotein, total bilirubin, direct bilirubin, and indirect bilirubin levels, and alkaline phosphatase, δ- glutamyl transferase, lactate dehyrogenase, aspartate aminotransferase, and alanine aminotransferase activities. In addition, histopathology of the liver of all the animals was evaluated. According to the obtained findings, the aflatoxin given at specified doses and period caused the extreme damage to the liver and sodium bentonite was effective to relieve this damage. Furthermore, it was demonstrated that total protein, δ- glutamyl trasferase, alanine aminotransferase, albumins, triglycerides, cholesterol, high-density lipoproteins, lowdensity lipoproteins, and total bilirubin levels/activity may be taken into consideration at the first place in the case of in vivo efficacy trials of binding agents against aflatoxins. Key words: broilers, aflatoxin, sodium bentonite. Aflatoxins are toxic metabolites of Aspergillus parasiticus and A. flavus, which can occur widely in poultry feeds and feedstuffs (3, 4). The levels of oxygen, ph, carbon dioxide, and moisture play a key role in the synthesis of these metabolites (4). Under optimum conditions, o-methylsterigmatocystin and dihydro-omethylsterigmatocystin are formed by acetate and malonyl-coenzyme A as a result of the series of enzymatic reactions developed in the fungi (3, 4). These compounds are intermediate metabolites responsible for the formation of B 1, B 2, G 1, and G 2 aflatoxins. Poultry are highly sensitive to these toxic compounds. Most conspicuous poisoning effects observed in poultry is damage to the liver, growth retardation; decreased feed intake and weakening of the immune system (3, 4, 6-12, 15). This situation brings into attention the necessary measures for minimizing these effects. The incorporating of binding agents added to feed at certain rates is one of the most commonly used measures (6, 8, 10, 11, 15). These binding agents have adsorbent structure and contain aluminosilicate (as well as sodium or calcium). Sodium and calcium, which are present in the chemical structures of these compounds, increase their binding capacity. Consequently, an irreversible structure is formed in the digestive tract as a result of interaction between binding agent and aflatoxins; and the absorption of aflatoxins is limited (14). There are numerous studies on the effects of binding agents against aflatoxins (6, 8, 10, 11, 13). The present study was intended to define whether aflatoxin given to chickens at the specified dose and period leads to any adverse effects or not and whether sodium bentonite is effective against aflatoxin poisoning. It will be also interesting which of the basic parameters that were applied to asses the efficiency of aflatoxin binding agents could be used in in vivo investigations.

2 108 Material and Methods Seventy-two one-day-old, male chicks were used. The chicks were weighed and divided into 6 equal groups. While the first (control) group for 45 d was fed on normal ration, at the same time the second, third, fourth, fifth, and sixth groups received feed containing, 0.25% sodium bentonite, 0.50% sodium bentonite, 1 ppm of aflatoxin (AF), 1 ppm of AF+0.25% sodium bentonite and 1 ppm of AF+0.50% sodium bentonite, respectively. At the end of the experimental period, the animals were sacrificed and the liver was cut out. Total protein, albumin, glucose, triglyceride, cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), total bilirubin, direct bilirubin, and indirect bilurubin concentrations, and alkaline phosphatase (ALP), δ- glutamyl transferase (GGT), lactate dehydrogenase (LDH), aspartate amino transferase (AST) and alanine amino transferase (ALT) activities were measured in the blood. Abbott kit and autoanalyser were used in biochemical analysis. The histopathological findings of the liver were evaluated (18). AF was produced, according to Shotwell et al. (22) method, which is based on the method of Demet et al. (5). Total AF level was measured by enzyme immunoassay apparatus, using r- biofarm kit and method suggested by the producer. The AF compounds, constituting total AF, were detected according to the method of Nabney and Nesbitt (19). Their approximate rates were 85%, 10%, 3%, and 2% for AF B 1, B 2, G 1, and G 2, The data were expressed as arithmetic means and standard deviation. One-way variance analysis (ANOVA) and Duncan test were used for statistical analysis. Results At the end of the experiment; significant decreases in the concentrations of total protein in groups IV and V (P<0.05), albumins in group IV (P<0.05), glucose in groups III and VI (P<0.05), cholesterol in groups II, IV, V, and VI (P<0.05), HDL in groups IV and V (P<0.05), LDL in groups II, III, IV, V, and VI (P<0.05), and of VLDL in groups III, IV, and VI (P<0.05), as compared to control group, were found (Tables 1 and 2). A significant increase in triglyceride level in group II (P<0.05) and a significant decrease in groups IV and VI (P<0.05), a significant decrease in GGT activity in groups IV and V (P<0.05), a significant increase in LDH activity in groups II, III, IV, and V (P<0.05), and AST activity in groups IV, V, and VI (P<0.05), a significant increase in ALT activity in group IV (P<0.05), and significant decrease in groups V and VI (P<0.05) were noted (Tables 1 and 4). Total bilirubin content was increased significantly in groups IV, V, and VI (P<0.05) and a significant increase in direct bilirubin level was observed in group IV (P<0.05) (Table 3). Histopathological examination demonstrated fatty degeneration of hepatocytes, mononuclear cell infiltration in periportal region, hyperplastic bile ducts, and hepatocellular degeneration in the liver of the group which received AF alone (group IV). Although these lesions were observed also in the liver of groups receiving AF+sodium bentonite combination (groups V and VI), they were not as severe as those in group IV (Figs 1-2). Table 1 Mean concentrations of total protein, albumins, glucose and triglycerides in control and trial groups Group* T-Protein (g/dl) Albumins (g/dl) Glucose Triglycerides Group I 3.11±0.12 b 1.48±0.09 a ±11.26 a 58.60±8.96 b Group II 2.96±0.14 b 1.36±0.10 a ±19.44 a 66.80±6.14 a Group III 3.01±0.13 b 1.43±0.09 a ±11.58 cd 53.40±5.02 b Group IV 2.19±0.06 a 1.15±0.06 b ±11.71 bc 43.40±4.33 c Group V 2.39±0.10 a 1.22±0.05 ab ±4.06 d 51.40±3.43 b Group VI 3.00±0.52 b 1.42±0.26 a ±9.71b c 47.66±2.08 c a, b, c, d Differences between means within the same column with different letters are statistically significant (P<0.05). * Group I-control; group II-sodium bentonite; group III-sodium bentonite; group IV- AF; group V-AF+sodium bentonite; group VI- AF+sodium bentonite. Table 2 Mean levels of cholesterol, HDL, LDL and VLDL in control and trial groups Group Cholesterol HDL LDL VLDL Group I ±13.70 a 50.00±4.24 a 50.00±2.73 a 11.82±1.47 ab Group II 92.80±4.76 b 46.40±4.09 ab 34.40±2.07 c 12.92±2.54 a Group III ±5.85 a 45.80±3.03 ab 42.40±5.81 b 9.24±1.74 c Group IV 57.00±5.95 d 37.40±1.51 c 15.60±0.54 e 7.90±0.52 cd Group V 78.20±7.19 c 42.20±2.77 bc 22.60±3.04 d 9.66±0.64 abc Group VI 88.66±6.65 bc 49.66±10.06 a 24.00±4.58 d 9.80±1.85 d a, b, c, d Differences between means within the same column with different letters are statistically significant (P<0.05).

3 109 Fig. 1. Fatty degeneration in hepatocytes. H. E., 20x (a); the infiltration of mononuclear cells in portal area. H. E., 20x (b); hyperplasia of bile ducts and extensive hepatocellular degeneration. H. E., 40x (c); extensive hepatocellular degeneration. H. E.; 40x (d). Fig. 2. Moderate affected hepatocytes and infiltration of mononuclear cells in broilers receiving low (e) and high (f) doses of bentonite. H. E.; 10x.

4 110 Table 3 Activity/level of TBIL, DBIL, INBIL and ALP in control and trial groups Group TBIL DBIL INBIL ALP Group I 0.38±0.05 a 0.04±0.04 a 0.33± ± Group II 0.37±0.04 a 0.05±0.01 a 0.32± ± Group III 0.35±0.04 a 0.06±0.02 ab 0.26± ± Group IV 0.48±0.02 b 0.13±0.03 b 0.30± ± Group V 0.42±0.01 c 0.11±0.03 ab 0.33± ± Group VI 0.40±0.02 c 0.11±0.01 ab 0.30± ± a, b, c Differences between means within the same column with different letters are statistically significant (P<0.05). Table 4 Activity of GGT, LDH, AST and ALT in control and trial groups Group GGT LDH AST ALT Group I 30.60±5.72 b ±69.19 b ±10.18 c 5.40±1.14 a Group II 28.20±4.71 ab ±29.27 a ±16.20 bc 4.40±1.67 a Group III 24.00±5.19 ab ± a ±52.71 abc 5.80±1.92 ab Group IV 14.80±6.30 c ±94.76 a ±28.90 ab 7.80±3.03 c Group V 21.00±4.79 a ± a ±28.70 ab 3.40±0.54 b Group VI 25.33±5.68 ab ± ab ±65.04 a 4.33±0.57 b a, b, c Differences between means within the same column with different letters are statistically significant (P<0.05). Discussion In this research, while the total protein levels of control group and that which received only sodium bentonite were very close to each other, a statistically significant decrease in total protein level of the group receiving only AF (group IV) was observed. This decrease, though less evident, was also observed in the group which received AF+sodium bentonite combination (groups V and VI). The results indicated that the liver was affected obviously by AF at the specified dose and period. This was observed clearly by the decrease in total protein level. On the other hand, it was noted that the adsorbent incorporated into feed alone, did not make any changes of this parameter, consequently, it did not have any adverse effect on the relevant organ as well. The changes observed in albumin levels were similar to those in total protein levels. Kececi et al. (16) and Oguz et al. (20) reported that AF declined the protein level and the adsorbents (clinoptilolite, polyvinylpolypyrrolidone, zeolite and bentonite) reduced its effect, regarding that parameter. In addition, a statistically significant decrease was observed in blood cholesterol level in the group which received only AF (group IV). This indicated that the liver was affected progressively by AF. In addition, the bile duct hyperplasia, hepatocellular degeneration, fatty changes in hepatocytes, and mononuclear cell infiltration observed in the histopathological examination of the liver of the group that received only AF, supported this conclusion. Ortatatli and Oguz (21) reported that there were similar pathological findings in the liver of broilers with aflatoxicosis. In the groups that received adsorbent+af combination, a statistically significant increase in cholesterol level, in connection with the given dose of the adsorbent, was observed. The changes in LDL, HDL, and VHLD levels in the control and trial groups confirmed the findings. Furthermore, Ledaux et al. (17) obtained the similar results in poultry. A statistically significant decrease was observed in GGT activity in the group that received AF alone (group IV). Aravind et al. (1) reported that AF caused changes of GGT activity in broilers. Only this decrease proved clearly that the liver was affected by AF. Firstly, the GGT activity in the groups V and VI remained higher and the difference in relation to the group IV was statistically significant. This indicated that the adsorbent was effective. Secondly, in these trials, an increase in ALP activity was observed in the group which received only AF, compared to controls, but was not statistically significant. Lastly, no significant changes were observed, regarding the total bilirubin level, in the groups which received only adsorbent. On the other hand, the significant increase was detected in the group which received AF alone. This increase is an indicator of malfunction in the liver, which resulted from AF action and, as explained above, probably this increase was related to the changes located specifically in bile ducts and hepatocytes. This was also supported by the histopathological findings. Furthermore, the statistically significant decrease in total bilirubin level was observed in the group that received AF with absorbent, when compared to the group that received AF alone. This decrease showed that AF was bound by the adsorbent. It was possible to see similar findings regarding the direct bilirubin, but not indirect bilirubin

5 111 levels. Awadalla (2) reported that AFled to an increase in blood bilirubin level in quails. A decrease in glucose level in all trial groups, compared to controls, was seen. These changes were much more critical in the group which received both AF and adsorbent (groups V and VI). The data were far from evaluated efficiency of binding agents against AF. The occurrence of the decrease only in the groups which received AF, indicated that the liver was damaged. Harvey et al. (13) reported that the blood glucose levels in animals that received AF declined and the administration of adsorbents with AF improved this parameter. Furthermore, a decrease in triglyceride concentration was detected in the groups which received AF+sodium bentonite.. But, this decrease was not as fast as that in the group which received only AF. These results indicated that AF affected this parameter and the rapid transporting process of lipids to the liver had an important role in the AF influence. The statistically significant increase in AST activity was observed in the groups which received AF alone and AF+sodium bentonite combination, compared to control. Aravind et al. (1) reported that AF increased AST and ALT activities. This increase clearly indicated the damage to the liver. In addition, the findings obtained by histopathological examination supported this conclusion as well. On the other hand, in the groups which received the adsorbent with AF (groups of V and VI), the increase was detected in the activity of ALT, when compared to the group which received AF alone. This situation clarified the efficiency of binding agent. The changes were similar to those observed in LDH activity. In addition, statistically insignificant differences regarding AST and ALT activities were shown between the control and group, which received sodium bentonite alone. In conclusion, AF given at the dose of 1 ppm for 45 d caused the extreme damage to the liver. This damage was obvious from the changes in biochemical parameters and histopathological examination of the liver. It was demonstrated that dietary sodium bentonite was effective against AF, but, at certain doses, could not bind AF completely. The values of some parameters in the groups which received the adsorbent with AF were close to those in the groups which received AF alone. So, it was clear that these parameters were easily affected following the ingestion of even the free part of AF (it is a kind of AF) which was easily absorbed by the digestive tract. Therefore, it was considered that the evaluations of the activities of GGT and ALT, and levels of total protein, albumins, triglycerides, cholesterol, HDL, LDL, and total bilirubin would be the most accurate approach in the studies to be conducted to determine the binding efficiency of adsorbents, incorporated into feed, against AF. References 1. 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